We are studying the pathogenesis of viral hepatitis and the molecular basis for virulence and attenuation of these important pathogens. Hepatitis A. We have shown previously that virulence and attenuation are controlled principally by two genes: VP1/2A and 2C. However, attenuating mutations are strongly selected against in vivo, resulting in the emergence of virulent variants. This has important implications for the development of live attenuated hepatitis A vaccines. The pathogenesis of hepatitis A is also being studied in the chimpanzee model by microarray analysis. Both innate and adaptive responses have been recorded.[unreadable] Hepatitis B. In collaborative studies with Dr. Frank Chisari (Scripps Institute) we have studied in chimpanzees the mechanism by which the host clears a hepatitis B virus infection and the relationship of these mechanisms to clinical disease. We demonstrated that the clearance of the template for HBV synthesis, covalently closed circular HBV DNA, is eliminated from hepatocytes by non-cytolytic mechanisms mediated principally by interferon gamma in the liver. Elimination of residual hepatocytes containing HBV antigens is a later event that is mediated by cytolytic CD8 positive T cells and is temperally related to the hepatitis phase of the infection. The spectrum of virus-induced and immune response-related genes involved in hepatitis B were further studied by microarray analysis of intrahepatic messenger RNAs up-regulated and down-regulated during the course of hepatitis B infections in chimpanzees. Surprisingly, we could not detect evidence of an innate immune response to infection, suggesting that HBV can subvert the host immune response, but we did detect a strong adaptive immune response during the clearance phase of infection; this correlated with the inhibition of viral replication and removal of infected cells described above.[unreadable] Hepatitis C. The genetic heterogeneity of hepatitis C virus is believed to play an important role in its pathogenicity. We have previously examined this relationship by determining the genetic heterogeneity of HCV isolates that were recovered from patients who were infected following transfusion in order to study the early phase of infection and from patients undergoing interferon therapy in order to study changes during the later phase of chronic infection. Distinctive patterns of dynamic change in the sequence of viral clones during the first several weeks of infection were observed and these correlated with the outcome of infection. Similarly, the pattern of dynamic changes in sequence during interferon therapy was predictive of the outcome. These findings may be useful in predicting the outcome of therapy with interferon early in the course of treatment. More recently, we have extended these studies to an analysis of HCV sequences in infants and young children who were infected at birth. Although such perinatal transmissions are rare, they do offer an opportunity to study the interaction of the developing host immune response in an immunologically naive infant. Transmission of HCV from mother to infant often is characterized by virtually monoclonal viral infection initially, probably resulting from immunological selection by the mother?s immune system. As the infant mounts its own immunological response the genomic sequence becomes heterogeneous. Thus, the progression from monoclonal to polyclonal viral populations resembles that seen in chimpanzees experimentally infected with monoclonal HCV. Although considerable information has been gained from these longitudinal studies of patients, it is difficult to study the mechanisms of pathogenesis in such systems. Chimpanzees, which are the only animals other than man that are susceptible to infection with HCV, provide an experimental model for studying the interactions of the host and the virus in the pathogenesis of hepatitis C. Collaborative studies with Frank Chisari have demonstrated that, as in hepatitis B virus infections, in hepatitis C virus infections the cellular immune response plays an important role in noncytolytic down-regulation of viral replication and cytolytic removal of residual infected cells. These two mechanisms are sequential and overlapping and the former appears to be mediated by interferon gamma and the latter by CD8 positive cells and, perhaps by interferon gamma through its proinflammatory activity. These studies have also revealed that type 1 interferon (interferon alpha/beta)-activated antiviral proteins are expressed in response to the viral infections, but that HCV is resistant to the antiviral activity of this innate immune response. Microarray studies of the host immune responses to viral hepatitis and how the hepatitis viruses attempt to circumvent the responses, are yielding important information on pathogenesis of these diseases, and the studies are being extended to the other hepatitis viruses in order to delineate the comparative pathogenesis of these agents in a single host, the chimpanzee, which is the only non-human host that is acceptable to all human hepatitis viruses. Other studies of hepatitis C in chimpanzees have revealed that a little understood protein, the p7 polypeptide, is critical for infectivity and contains functionally important genotype-specific sequences. This finding provides another potential target for the development of antiviral agents against hepatitis C.[unreadable] Hepatitis D. Hepatitis D virus is a defective virus that requires coinfection with hepatitis B virus for its replication. Although infections with HDV are uncommon, they are associated with severe, rapidly progressing chronic hepatitis leading to death within a few years. In a long-term study of interferon alpha therapy of chronic hepatitis D, a remarkable regression of advanced hepatic cirrhosis was observed. Although reversal of cirrohsis has traditionally been thought not to be possible, this and other studies provide hope for severely affected patients, whose only recourse at this time is liver transplantation. Currently, we are also studying the pathogenesis of HDV infections by microarray analysis and have identified both innate and adaptive immune responses to such infections.[unreadable] Hepatitis E. Although rare in the United States, hepatitis E is the single most important cause of acute hepatitis among adults throughout Asia, the Middle East and North Africa. Like most of the hepatitis viruses, it replicates poorly or not at all in cell culture and cannot be transmitted to small laboratory animals. We have developed replicons for the study of HEV in vitro; these tools are permitting a detailed molecular analysis of viral replication that can be confirmed in vivo with molecularly engineered infectious cDNA clones of the virus. In addition, with colleagues, we are developing small animal models (swine HEV in swine, avian HEV in chickens) that, with nonhuman primate models of HEV, provide an unprecedented opportunity for studying the comparative pathogenesis of hepatitis E viruses. Finally, hepatitis E has also been studied by microarray and a brisk innate but weak adaptive immune response has been seen.